Human aggrecanase and nucleic acid compositions encoding the same

ABSTRACT

Human aggrecanase and polypeptides related thereto, as well as nucleic acid compositions encoding the same, are provided. The subject polypeptides and nucleic acid compositions find use in a variety of applications, including research, diagnostic, and therapeutic agent screening applications. Also provided are methods of inhibiting aggrecanase activity in a host and methods of treating disease conditions associated with aggrecanase activity, e.g. rheumatoid arthritis, osteo-arthritis, infectious arthritis, gouty arthritis, psoriatic arthritis, spondolysis, sports injury, joint trauma, pulmonary disease, fibrosis, and the like.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a divisional of U.S. patent application Ser. No.09/568,559 filed on May 9, 2000, now issued as U.S. Pat. No. 6,649,377,the disclosure of which is incorporated herein in its entirety. Thispatent claims the benefit under Title 35 U.S.C. 119(e) of U.S.Provisioual Applications Nos. 60/133,343 filed May 10, 1999, thedisclosure of which is incorporated herein in its entirety.

INTRODUCTION

1. Field of the Invention

The field of the invention is proteases, particularly proteases thatcleave aggrecan.

2. Background of the Invention

Cartilage matrix structure as dry weight of the tissue is made up of 70%collagen and 20–30% proteoglycans. The proteoglycan component confersmechanical flexibility to load bearing tissues and imparts viscoelasticproperties to cartilage. Its loss leads to rapid structural damage as isseen most frequently in arthritic joint diseases and joint injury.

Aggrecan is a major cartilage proteoglycan. Aggrecan is a large proteinof 210 kDa and has three globular domains: G1, G2, and G3. The G1 and G2domains of the protein are closer to the amino terminus of the proteinand their intervening interglobular domain has sites that areproteolytically sensitive. The region between G2 and G3 is heavilyglycosylated and connected to oligosaccharides and glycosaminoglycans(GAGs) to form the mature proteoglycan. In arthritic cartilage, coreprotein fragments of 55 kDa are observed and believed to be the resultof cleavage of the core protein in the G1 and G2 interglobular domainbetween asparagine 341 and phenylalanine 342. This cleavage can be madeby many matrix metalloproteinases e.g. MMP-1, -2, -3, -7, -8, -9, and-13. In addition, 60 kDa aggrecan fragments with a —COOH terminus ofglutamic acid are also identified and are indicative of a cleavage sitebetween glutamic acid 373 and alanine 374. Matrix metalloproteinase areunable to cleave at this site. The unique endopeptidase activityresponsible for this cleavage has been termed “aggrecanase.”

The G1 domain of the core protein forms a stable ternary complex bybinding to hyaluronic acid and link proteins in the matrix. Anyenzymatic cleavage in this region destabilizes the cartilage matrixstructure, leads to the loss of the major proteoglycan aggrecan andexposes type II collagen to collagenases, causing cartilage loss and theconsequent development of joint disease. Since a variety ofanti-arthritic drugs do not target aggrecanase and are incapable ofblocking cleavage of aggrecan, the aggrecanase site plays a key role inthe proteolytic degradation of aggrecan.

As such, aggrecanase is considered to be an important drug target forarthritis. Aggrecan fragments released into the synovial fluid are theprimary detectable events in the development of rheumatoid- andosteo-arthritis. Search for this protease has been intense. Despitethese intense discovery efforts, identification of human aggrecanase hasremained elusive.

As such, there is much interest in the identification of humanaggrecanase, as well as the gene encoding this activity.

Relevant Literature

U.S. Patents of interest include: U.S. Pat. Nos. 5,872,209 and5,427,954. PCT publications of interest include: WO 99/09000; WO98/55643; WO 98/51665; and WO 97/18207.

Other references of interest include: Vankemmelbeke et al.,“Coincubation of bovine synovial or capsular tissue with cartilagegenerates a soluble ‘Aggrecanase’ activity,” Biochem Biophys Res Commun(1999 Feb. 24) 255(3):686–91; Arner et al., “Generation andCharacterization of Aggrecanase. A soluble, cartilage-derivedaggrecan-degrading activity,” J Biol Chem (1999 Mar. 5)274(10):6594–6601; Billington et al., “An aggrecan-degrading activityassociated with chondrocyte membranes,” Biochem J (1998 Nov. 15) 336 (Pt1):207–12; Hughes et al., “Differential expression of aggrecanase andmatrix metalloproteinase activity in chondrocytes isolated from bovineandporcine articular cartilage,” J Biol Chem (1998 Nov 13)273(46):30576–82; Sandy et al., “Chondrocyte-mediated catabolism ofaggrecan: aggrecanase-dependent cleavage induced by interleukin-1 orretinoic acid can be inhibited by glucosamine,” Biochem J (1998 Oct. 1)335 (Pt 1):59–66; Amer et al., “Cytokine-induced cartilage proteoglycandegradation is mediated by aggrecanase,” Osteoarthritis Cartilage (1998May) 6(3):214–28; Ilic et al., “Characterization of aggrecan retainedand lost from the extracellular matrix of articular cartilage.Involvement of carboxyl-terminal processing in the catabolism ofaggrecan,” J Biol Chem (1998 Jul. 10) 273(28):17451–8; and Buttner etal., “Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves therecombinant aggrecan substrate rAgg1mut at the ‘aggrecanase’ and the MMPsites. Characterization of MT1-MMP catabolic activities on theinterglobular domain of aggrecan,” Biochem J (1998 Jul. 1)333 (Pt1):159–65.

SUMMARY OF THE INVENTION

Human aggrecanase and polypeptides related thereto, as well as nucleicacid compositions encoding the same, are provided. The subjectpolypeptide and nucleic acid compositions find use in a variety ofapplications, including research, diagnostic, and therapeutic agentscreening applications, as well as in treatment therapies. Also providedare methods of treating disease conditions associated with aggrecanaseactivity, e.g. conditions characterized by the presence of aggrecancleavage products, such as rheumatoid- and osteo-arthritis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the nucleic acid and amino acid sequence of humanaggrecanase.

DETAILED DESCRIPTION OF THE INVENTION

A novel human enzyme having aggrecan cleavage activity (i.e. humanaggrecanase or ADAMTS-1) and polypeptides related thereto, as well asnucleic acid compositions encoding the same, are provided. The subjectpolypeptide and/or nucleic acid compositions find use in a variety ofdifferent applications, including research, diagnostic, and therapeuticagent screening/discovery/preparation applications. Also provided aremethods of treating disease conditions associated with aggrecanasefunction, e.g. diseases characterized by the presence of aggrecancleavage products, such as rheumatoid- and osteo-arthritis.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Polypeptide Compositions

A novel human enzyme having aggrecanase activity (i.e. human aggrecanaseor ADAMTS-1), as well as polypeptide compositions related thereto, areprovided. The term polypeptide composition as used herein refers to boththe full length human protein, as well as portions or fragments thereof.Also included in this term are variations of the naturally occurringhuman protein, where such variations are homologous or substantiallysimilar to the naturally occurring protein, as described in greaterdetail below. In the following description of the subject invention, theterm “aggrecanase” is used to refer to the wild type human aggrecanasemolecule of the subject invention.

The aggrecanase protein of the subject invention is an enzyme,particularly a proteinase and more particularly a metalloproteinase. Thesubject human aggrecanase is characterized by having aggrecanaseactivity. As such, the subject aggrecanase is capable of cleavingaggrecan in an interglobular domain, particularly between the G1 and G2domains, and more particularly at the Glu³⁷³-Ala³⁷⁴ bond of humanaggrecan, to produce a cleavage product having an N-terminal sequence ofARGSVIL.

Human aggrecanase has an amino acid sequence as shown in FIG. 1 andidentified as SEQ ID NO:02. Human aggrecanase has a molecular weightbased on its amino acid sequence of about 100 kDa. The true molecularweight of human aggrecanase may vary due to glycosylation and/or otherpostranslational modifications. As such, the actual molecular weight ofhuman aggrecanase is likely to be in the range from about 80 to 100 kDa,usually from about 85 to 95 kDa.

Aggrecanase homologs or proteins (or fragments thereof) that vary insequence from the wild type sequence of the subject invention are alsoprovided. By homolog is meant a protein having at least about 35%,usually at least about 40% and more usually at least about 60% aminoacid sequence identity to the human aggrecanase protein of the subjectinvention, as determined using MegAlign, DNAstar (1998) clustalalgorithm as described in D. G. Higgins and P. M. Sharp, “Fast andSensitive multiple Sequence Alignments on a Microcomputer,” (1989)CABIOS, 5:151–153. (Parameters used are ktuple 1, gpa penalty 3, window,5 and diagonals saved 5).

Also provided are aggrecanase proteins that are substantially identicalto the hu aggrecanase protein, where by substantially identical is meantthat the protein has an amino acid sequence identity to the sequence ofaggrecanase of at least about 60%, usually at least about 65% and moreusually at least about 70%.

The proteins of the subject invention are present in a non-naturallyoccurring environment, e.g. are separated from their naturally occurringenvironment. In certain embodiments, the subject proteins are present ina composition that is enriched for the subject protein as compared toits naturally occurring environment. For example, purified aggrecanaseis provided, where by purified is meant that the aggrecanase enzyme ispresent in a composition that is substantially free of non-aggrecanaseproteins, where by substantially free is meant that less than 90%,usually less than 60% and more usually less than 50% of the compositionis made up of non-aggrecanase proteins. The proteins of the subjectinvention may also be present as an isolate, by which is meant that theprotein is substantially free of other proteins and other naturallyoccurring biologic molecules, such as oligosaccharides, polynucleotidesand fragments thereof, and the like, where the term “substantially free”in this instance means that less than 70%, usually less than 60% andmore usually less than 50% of the composition containing the isolatedprotein is some other naturally occurring biological molecule. Incertain embodiments, the proteins are present in substantially pureform, where by “substantially pure form” is meant at least 95%, usuallyat least 97% and more usually at least 99% pure.

In addition to the naturally occurring proteins, polypeptides which varyfrom the naturally occurring proteins are also provided, e.g.aggrecanase polypeptides. By aggrecanase polypeptide is meant an aminoacid sequence encoded by an open reading frame (ORF) of the geneencoding aggrecanase, described in greater detail below, including thefull length aggrecanase protein and fragments thereof, particularlybiologically active fragments and/or fragments corresponding tofunctional domains, e.g. transmembrane domain, and the like; andincluding fusions of the subject polypeptides to other proteins or partsthereof. Fragments of interest will typically be at least about 10 aa inlength, usually at least about 50 aa in length, and may be as long as300 aa in length or longer, but will usually not exceed about 1000 aa inlength, where the fragment will have a stretch of amino acids that isidentical to the subject protein of at least about 10 aa, and usually atleast about 15 aa, and in many embodiments at least about 50 aa inlength.

The subject proteins and polypeptides may be obtained from naturallyoccurring sources or synthetically produced. For example, humanaggrecanase may be derived from biological sources which expressaggrecanase, such as synoviocytes, chondrocytes, cartilege and the like.The subject proteins may also be derived from synthetic means, e.g. byexpressing a recombinant gene encoding protein of interest in a suitablehost, as described in greater detail below. Any convenient proteinpurification procedures may be employed, where suitable proteinpurification methodologies are described in Guide to ProteinPurification, (Deuthser ed.) (Academic Press, 1990). For example, alysate may prepared from the original source, e.g. chondrocytes or theexpression host, and purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, and the like.

Nucleic Acid Compositions

Also provided are nucleic acid compositions encoding aggrecanaseproteins or fragments thereof, as well as the aggrecanase homologues ofthe present invention. By aggrecanase nucleic acid composition is meanta composition comprising a sequence of DNA having an open reading framethat encodes aggrecanase, i.e. an aggrecanase gene, and is capable,under appropriate conditions, of being expressed as aggrecanase. Alsoencompassed in this term are nucleic acids that are homologous orsubstantially similar or identical to the nucleic acids encodingaggrecanase proteins. Thus, the subject invention provides genesencoding the human aggrecanase of the subject invention and homologsthereof. The human aggrecanase gene has the nucleic acid sequence shownin FIG. 1 and identified as SEQ ID NO:01, infra.

The source of homologous genes may be any species, e.g., primatespecies, particularly human; rodents, such as rats and mice, canines,felines, bovines, ovines, equines, yeast, nematodes, etc. Betweenmammalian species, e.g., human and mouse, homologs have substantialsequence similarity, e.g. at least 75% sequence identity, usually atleast 90%, more usually at least 95% between nucleotide sequences.Sequence similarity is calculated based on a reference sequence, whichmay be a subset of a larger sequence, such as a conserved motif, codingregion, flanking region, etc. A reference sequence will usually be atleast about 18 nt long, more usually at least about 30 nt long, and mayextend to the complete sequence that is being compared. Algorithms forsequence analysis are known in the art, such as BLAST, described inAltschul et al. (1990), J. Mol. Biol. 215:403–10 (using defaultsettings, i.e. parameters w=4 and T=17). The sequences provided hereinare essential for recognizing aggrecanase-related and homologousproteins, and the nucleic acids encoding the same, in database searches.

Nucleic acids encoding the aggrecanase protein and aggrecanasepolypeptides of the subject invention may be cDNA or genomic DNA or afragment thereof. The term “aggrecanase gene” shall be intended to meanthe open reading frame encoding specific aggrecanase proteins andpolypeptides, and aggrecanase introns, as well as adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression, up to about 20 kb beyond the coding region, but possiblyfurther in either direction. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto a host genome.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 5′ and 3′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding anaggrecanase protein.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include 5′ and 3′ un-translatedregions found in the mature mRNA. It may further include specifictranscriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The nucleic acid compositions of the subject invention may encode all ora part of the subject aggrecanase protein. Double or single strandedfragments may be obtained from the DNA sequence by chemicallysynthesizing oligonucleotides in accordance with conventional methods,by restriction enzyme digestion, by PCR amplification, etc. For the mostpart, DNA fragments will be of at least 15 nt, usually at least 18 nt or25 nt, and may be at least about 50 nt.

The aggrecanase genes are isolated and obtained in substantial purity,generally as other than an intact chromosome. Usually, the DNA will beobtained substantially free of other nucleic acid sequences that do notinclude an aggrecanase sequence or fragment thereof, generally being atleast about 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

Preparation of Aggrecanase Polypeptides

In addition to the plurality of uses described in greater detail infollowing sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the aggrecanase polypeptides, asdescribed above. The provided polynucleotide (e.g., a polynucleotidehaving a sequence of SEQ ID NO:01, the corresponding cDNA, or thefull-length gene is used to express a partial or complete gene product.Constructs of polynucleotides having a sequences of “SEQ ID NO:01 can begenerated synthetically. Alternatively, single-step assembly of a geneand entire plasmid from large numbers of oligodeoxyribonucleotides isdescribed by, e.g., Stemmer et al., Gene (Amsterdam) (1995)164(1):49–53. In this method, assembly PCR (the synthesis of long DNAsequences from large numbers of oligodeoxyribonucleotides (oligos)) isdescribed. The method is derived from DNA shuffling (Stemmer, Nature(1994) 370:389–391), and does not rely on DNA ligase, but instead relieson DNA polymerase to build increasingly longer DNA fragments during theassembly process. Appropriate polynucleotide constructs are purifiedusing standard recombinant DNA techniques as described in, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989)Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and under currentregulations described in United States Dept. of HHS, National Instituteof Health (NIH) Guidelines for Recombinant DNA Research.

Polynucleotide molecules comprising a polynucleotide sequence providedherein are propagated by placing the molecule in a vector. Viral andnon-viral vectors are used, including plasmids. The choice of plasmidwill depend on the type of cell in which propagation is desired and thepurpose of propagation. Certain vectors are useful for amplifying andmaking large amounts of the desired DNA sequence. Other vectors aresuitable for expression in cells in culture. Still other vectors aresuitable for transfer and expression in cells in a whole animal orperson. The choice of appropriate vector is well within the skill of theart. Many such vectors are available commercially. The partial orfull-length polynucleotide is inserted into a vector typically by meansof DNA ligase attachment to a cleaved restriction enzyme site in thevector. Alternatively, the desired nucleotide sequence can be insertedby homologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

For expression, an expression cassette or system may be employed. Thegene product encoded by a polynucleotide of the invention is expressedin any convenient expression system, including, for example, bacterial,yeast, insect, amphibian and mammalian systems. Suitable vectors andhost cells are described in U.S. Pat. No. 5,654,173. In the expressionvector, an aggrecanase encoding polynucleotide, e.g. as set forth in SEQID NO:01, is linked to a regulatory sequence as appropriate to obtainthe desired expression properties. These can include promoters (attachedeither at the 5′ end of the sense strand or at the 3′ end of theantisense strand), enhancers, terminators, operators, repressors, andinducers. The promoters can be regulated or constitutive. In somesituations it may be desirable to use conditionally active promoters,such as tissue-specific or developmental stage-specific promoters. Theseare linked to the desired nucleotide sequence using the techniquesdescribed above for linkage to vectors. Any techniques known in the artcan be used. In other words, the expression vector will provide atranscriptional and translational initiation region, which may beinducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.These control regions may be native to the aggrecanase gene, or may bederived from exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. β-galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

Aggrecanase proteins and polypeptides may be expressed in prokaryotes oreukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, HEK 293, CHO, Xenopus Oocytes, etc., may be used as theexpression host cells. In some situations, it is desirable to expressthe aggrecanase gene in eukaryotic cells, where the aggrecanase proteinwill benefit from native folding and post-translational modifications.Small peptides can also be synthesized in the laboratory. Polypeptidesthat are subsets of the complete aggrecanase sequence may be used toidentify and investigate parts of the protein important for function.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21–25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284–289;Tilburn et al., Gene (1983) 26:205–221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470–1474; Kelly and Hynes, EMBO J. (1985)4:475479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765–776; Miller etal., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda et al., Nature (1985) 315:592–594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47–55, Miller etal., Generic Engineering (1986) 8:277–279, and Maeda et al., Nature(1985) 315:592–594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. RE 30,985.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism. The product isrecovered by any appropriate means known in the art.

Once the gene corresponding to a selected polynucleotide is identified,its expression can be regulated in the cell to which the gene is native.For example, an endogenous gene of a cell can be regulated by anexogenous regulatory sequence inserted into the genome of the cell atlocation sufficient to at least enhance expressed of the gene in thecell. The regulatory sequence may be designed to integrate into thegenome via homologous recombination, as disclosed in U.S. Pat. Nos.5,641,670 and 5,733,761, the disclosures of which are hereinincorporated by reference, or may be designed to integrate into thegenome via non-homologous recombination, as described in WO 99/15650,the disclosure of which is herein incorporated by reference. As such,also encompassed in the subject invention is the production of thesubject aggrecanase proteins without manipulation of the encodingnucleic acid itself, but instead through integration of a regulatorysequence into the genome of cell that already includes a gene encodingthe desired protein, as described in the above incorporated patentdocuments.

Uses of the Subject Aggrecanase Polypeptide and Nucleic AcidCompositions

The subject polypeptide and nucleic acid compositions find use in avariety of different applications, including research, diagnostic, andtherapeutic agent screening/discovery/preparation applications, as wellas in therapeutic compositions and methods employing the same.

Research Applications

The subject nucleic acid compositions find use in a variety of researchapplications. Research applications of interest include: theidentification of aggrecanase homologs; as a source of novel promoterelements; the identification of aggrecanase expression regulatoryfactors; as probes and primers in hybridization applications, e.g. PCR;the identification of expression patterns in biological specimens; thepreparation of cell or animal models for aggrecanase function; thepreparation of in vitro models for aggrecanase function; etc.

Homologs of the aggrecanase gene are identified by any of a number ofmethods. A fragment of the provided cDNA may be used as a hybridizationprobe against a cDNA library from the target organism of interest, wherelow stringency conditions are used. The probe may be a large fragment,or one or more short degenerate primers. Nucleic acids having sequencesimilarity are detected by hybridization under low stringencyconditions, for example, at 50° C. and 6×SSC (0.9 M sodium chloride/0.09M sodium citrate) and remain bound when subjected to washing at 55° C.in 1×SSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequenceidentity may be determined by hybridization under stringent conditions,for example, at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5mM sodium citrate). Nucleic acids having a region of substantialidentity to the provided aggrecanase sequences, e.g. allelic variants,genetically altered versions of the gene, etc., bind to the providedaggrecanase sequences under stringent hybridization conditions. By usingprobes, particularly labeled probes of DNA sequences, one can isolatehomologous or related genes.

The sequence of the 5′ flanking region may be utilized for promoterelements, including enhancer binding sites, that provide fordevelopmental regulation in tissues where aggrecanase is expressed. Thetissue specific expression is useful for determining the pattern ofexpression, and for providing promoters that mimic the native pattern ofexpression. Naturally occurring polymorphisms in the promoter region areuseful for determining natural variations in expression, particularlythose that may be associated with disease.

Alternatively, mutations may be introduced into the promoter region todetermine the effect of altering expression in experimentally definedsystems. Methods for the identification of specific DNA motifs involvedin the binding of transcriptional factors are known in the art, e.g.sequence similarity to known binding motifs, gel retardation studies,etc. For examples, see Blackwell et al. (1995), Mol. Med. 1:194–205;Mortlock et al. (1996), Genome Res. 6:327–33; and Joulin and Richard-Foy(1995), Eur. J. Biochem. 232:620–626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of aggrecanasegene expression, especially in different tissues or stages ofdevelopment, and to identify cis acting sequences and trans-actingfactors that regulate or mediate aggrecanase gene expression. Suchtranscription or translational control regions may be operably linked toan aggrecanase gene in order to promote expression of wild type oraltered aggrecanase or other proteins of interest in cultured cells, orin embryonic, fetal or adult tissues, and for gene therapy.

Small DNA fragments are useful as primers for PCR, hybridizationscreening probes, etc. Larger DNA fragments, i.e. greater than 100 ntare useful for production of the encoded polypeptide, as described inthe previous section. For use in geometric amplification reactions, suchas geometric PCR, a pair of primers will be used. The exact compositionof the primer sequences is not critical to the invention, but for mostapplications the primers will hybridize to the subject sequence understringent conditions, as known in the art. It is preferable to choose apair of primers that will generate an amplification product of at leastabout 50 nt, preferably at least about 100 nt. Algorithms for theselection of primer sequences are generally known, and are available incommercial software packages. Amplification primers hybridize tocomplementary strands of DNA, and will prime towards each other.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of aggrecanase gene expression in the sample.

The sequence of an aggrecanase gene, including flanking promoter regionsand coding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or protein product of such a mutationwill usually be substantially similar to the sequences provided herein,i.e. will differ by at least one nucleotide or amino acid, respectively,and may differ by at least two but not more than about ten nucleotidesor amino acids. The sequence changes may be substitutions, insertions,deletions, or a combination thereof. Deletions may further includelarger changes, such as deletions of a domain or exon. Othermodifications of interest include epitope tagging, e.g. with the FLAGsystem, HA, etc. For studies of subcellular localization, fusionproteins with green fluorescent proteins (GFP) may be used.

Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for site specific mutagenesis may be found in Gustin et al.(1993), Biotechniques 14:22; Barany (1985), Gene 37:111–23; Colicelli etal. (1985), Mol. Gen. Genet. 199:537–9; and Prentki et al. (1984), Gene29:303–13. Methods for site specific mutagenesis can be found inSambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 15.3–15.108; Weiner et al. (1993), Gene 126:35–41; Sayers et al.(1992), Biotechniques 13:592–6; Jones and Winistorfer (1992),Biotechniques 12:528–30; Barton et al. (1990), Nucleic Acids Res18:7349–55; Marotti and Tomich (1989), Gene Anal. Tech. 6:67–70; and Zhu(1989), Anal Biochem 177:120–4. Such mutated genes may be used to studystructure-function relationships of aggrecanase, or to alter propertiesof the protein that affect its function or regulation.

The subject nucleic acids can be used to generate transgenic, non-humananimals or site specific gene modifications in cell lines. Transgenicanimals may be made through homologous recombination, where theendogenous aggrecanase locus is altered. Alternatively, a nucleic acidconstruct is randomly integrated into the genome. Vectors for stableintegration include plasmids, retroviruses and other animal viruses,YACs, and the like.

The modified cells or animals are useful in the study of aggrecanasefunction and regulation. Of interest is the use of aggrecanase genes toconstruct transgenic animal models of aggrecanase related diseaseconditions, e.g. disease conditions associated with aggrecanaseactivity, such as arthritis. Thus, transgenic animal models of thesubject invention include endogenous aggrecanase knockouts in whichexpression of endogenous aggrecanase is at least reduced if noteliminated, where such animals also typically express an aggrecanasepeptide of the subject invention, e.g. the aggrecanase protein of thesubject invention or a fragment thereof. Where a nucleic acid having asequence found in the human aggrecanase gene is introduced, theintroduced nucleic acid may be either a complete or partial sequence ofthe aggrecanase gene. A detectable marker, such as lac Z may beintroduced into the aggrecanase locus, where upregulation of aggrecanaseexpression will result in an easily detected change in phenotype. Onemay also provide for expression of the aggrecanase gene or variantsthereof in cells or tissues where it is not normally expressed, atlevels not normally present in such cells or tissues.

DNA constructs for homologous recombination will comprise at least aportion of the aggrecanase gene of the subject invention, wherein thegene has the desired genetic modification(s), and includes regions ofhomology to the target locus. DNA constructs for random integration neednot include regions of homology to mediate recombination. Conveniently,markers for positive and negative selection are included. Methods forgenerating cells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol.185:527–537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on aggrecanase activity.

Diagnostic Applications

Also provided are methods of diagnosing disease states based on observedlevels of aggrecanase or the expression level of the aggrecanase gene ina biological sample of interest. Samples, as used herein, includebiological fluids such as blood, cerebrospinal fluid, tears, saliva,lymph, dialysis fluid, and the like; organ or tissue culture derivedfluids; and fluids extracted from physiological tissues. Also includedin the term are derivatives and fractions of such fluids. The cells maybe dissociated, in the case of solid tissues, or tissue sections may beanalyzed. Alternatively a lysate of the cells may be prepared.

A number of methods are available for determining the expression levelof a gene or protein in a particular sample. Diagnosis may be performedby a number of methods to determine the absence or presence or alteredamounts of normal or abnormal aggrecanase in a patient sample. Forexample, detection may utilize staining of cells or histologicalsections with labeled antibodies, performed in accordance withconventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies of interest are added to the cell sample, andincubated for a period of time sufficient to allow binding to theepitope, usually at least about 10 minutes. The antibody may be labeledwith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabels for direct detection. Alternatively, a second stage antibody orreagent is used to amplify the signal. Such reagents are well known inthe art. For example, the primary antibody may be conjugated to biotin,with horseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to afluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

Alternatively, one may focus on the expression of aggrecanase.Biochemical studies may be performed to determine whether a sequencepolymorphism in an aggrecanase coding region or control regions isassociated with disease. Disease associated polymorphisms may includedeletion or truncation of the gene, mutations that alter expressionlevel, that affect the activity of the protein, etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of aggrecanase can be compared to expression levels of the normalallele by various methods known in the art. Methods for determiningpromoter or enhancer strength include quantitation of the expressednatural protein; insertion of the variant control element into a vectorwith a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence, e.g. a disease associated polymorphism.Where large amounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. Cells that expressaggrecanase may be used as a source of mRNA, which may be assayeddirectly or reverse transcribed into cDNA for analysis. The nucleic acidmay be amplified by conventional techniques, such as the polymerasechain reaction (PCR), to provide sufficient amounts for analysis. Theuse of the polymerase chain reaction is described in Saiki, et al.(1985), Science 239:487, and a review of techniques may be found inSambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 14.2–14.33. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1990), Nucl. Acids Res. 18:2887–2890; andDelahunty et al. (1996), Am. J. Hum. Genet. 58:1239–1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a wild-type aggrecanase gene sequence. Hybridization withthe variant sequence may also be used to determine its presence, bySouthern blots, dot blots, etc. The hybridization pattern of a controland variant sequence to an array of oligonucleotide probes immobilizedon a solid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease, the sampleis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

Screening for mutations in aggrecanase may be based on the functional orantigenic characteristics of the protein. Protein truncation assays areuseful in detecting deletions that may affect the biological activity ofthe protein. Various immunoassays designed to detect polymorphisms inaggrecanase proteins may be used in screening. Where many diversegenetic mutations lead to a particular disease phenotype, functionalprotein assays have proven to be effective screening tools. The activityof the encoded aggrecanase protein may be determined by comparison withthe wild-type protein.

Diagnostic methods of the subject invention in which the level ofaggrecanase gene expression is of interest will typically involvecomparison of the aggrecanase nucleic acid abundance of a sample ofinterest with that of a control value to determine any relativedifferences, where the difference may be measured qualitatively and/orquantitatively, which differences are then related to the presence orabsence of an abnormal aggrecanase gene expression pattern. A variety ofdifferent methods for determine the nucleic acid abundance in a sampleare known to those of skill in the art, where particular methods ofinterest include those described in: Pietu et al., Genome Res. (June1996) 6: 492–503; Zhao et al., Gene (Apr. 24, 1995) 156: 207–213;Soares, Curr. Opin. Biotechnol. (October 1997) 6: 542–546; Raval, J.Pharmacol Toxicol Methods (November 1994) 32: 125–127; Chalifour et al.,Anal. Biochem (Feb. 1, 1994) 216: 299–304; Stolz & Tuan, Mol.Biotechnol. (December 19960 6: 225–230; Hong et al., Bioscience Reports(1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143: 298. Also ofinterest are the methods disclosed in WO 97/27317, the disclosure ofwhich is herein incorporated by reference.

Screening Assays

The subject aggrecanase polypeptides find use in various screeningassays designed to identify therapeutic agents. In vitro screeningassays can be employed in which the activity of aggrecanase is assessedin the presence of a candidate therapeutic agent and compared to acontrol, i.e. the activity in the absence of the candidate therapeuticagent. Activity can be determined in a number of different ways, whereactivity may generally be determined as ability to cleave aggrecan or atleast a fragment therefore, as well as a recombinant polypeptide, thatincludes the aggrecanase cleavage site, as described above. Such assaysare described in U.S. Pat. No. 5,872,209 and WO 99/05921, thedisclosures of which are herein incorporated by reference, as well asArner et al., J. Biol. Chem. (March 1999) 274: 6594–6601.

Also of interest in screening assays are non-human transgenic animalswhich express functional aggrecanase, where such animals are describedabove. In many embodiments, the animals lack endogenous aggrecanase. Inusing such animals for screening applications, a test compound(s) isadministered to the animal, and the resultant changes in phenotype, e.g.presence of aggrecan produced by cleavage of the Glu³⁷³-Ala³⁷⁴ bond, arecompared with a control.

Alternatively, in vitro models of aggrecanase binding activity may bemeasured in which binding events between aggrecanase and candidateaggrecanase modulatory agents are monitored.

A variety of other reagents may be included in the screening assays,depending on the particular screening protocols employed. These includereagents like salts, neutral proteins, e.g. albumin, detergents, etcthat are used to facilitate optimal protein-protein binding and/orreduce non-specific or background interactions. Reagents that improvethe efficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc. may be used.

A variety of different candidate therapeutic agents that serve as eitheraggrecanase agonists or antagonists may be screened by the abovemethods. Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Of particular interest in many embodiments are screening methods thatidentify agents that selectively modulate, e.g. inhibit, the subjectaggrecanase enzyme and not other proteases.

Aggrecanase Nucleic Acid and Polypeptide Therapeutic Compositions

The nucleic acid compositions of the subject invention also find use astherapeutic agents in situations where one wishes to enhance aggrecanaseactivity in a host. The aggrecanase genes, gene fragments, or theencoded aggrecanase protein or protein fragments are useful in genetherapy to treat disorders associated with aggrecanase defects.Expression vectors may be used to introduce the aggrecanase gene into acell. Such vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences. Transcription cassettes may be prepared comprising atranscription initiation region, the target gene or fragment thereof,and a transcriptional termination region. The transcription cassettesmay be introduced into a variety of vectors, e.g. plasmid; retrovirus,e.g. lentivirus; adenovirus; and the like, where the vectors are able totransiently or stably be maintained in the cells, usually for a periodof at least about one day, more usually for a period of at least aboutseveral days to several weeks.

The gene or aggrecanase protein may be introduced into tissues or hostcells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992),Anal Biochem 205:365–368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al (1992), Nature 356:152–154), where gold microprojectiles arecoated with the DNA, then bombarded into skin cells.

Methods of Modulating Aggrecanase Activity

The subject invention provides methods of modulating aggrecanaseactivity in a cell, including methods of increasing aggrecanase activity(e.g. methods of enhancing), as well as methods of reducing orinhibiting aggrecanase activity, e.g. methods of stopping or limitingaggrecan cleavage. In such methods, an effective amount of anaggrecanase modulatory agent is contacted with the cell.

Also provided are methods of modulating, including enhancing andinhibiting, aggrecanase activity in a host. In such methods, aneffective amount of active agent that modulates the activity ofaggrecanase in vivo, e.g. usually enhances or inhibits aggrecanaseactivity, is administered to the host. The active agent may be a varietyof different compounds, including a naturally occurring or syntheticsmall molecule compound, an antibody, fragment or derivative thereof, anantisense composition, and the like.

Of particular interest in certain embodiments are agents that reduceaggrecanase activity, e.g. aggrecan cleavage, by at least about 10 fold,usually at least about 20 fold and more usually at least about 25 fold,as measure by the Assay described in Amer et al. (1999), supra. In manyembodiments, of particular interest is the use of compounds that reduceaggrecanase activity by at least 100 fold, as compared to a control.

Also of interest is the use of agents that, while providing for reducedaggrecanase activity, do not substantially reduce the activity of otherproteinases, if at all. Thus, the agents in this embodiment areselective inhibitors of aggrecanase. An agent is considered to beselective if it provides for the above reduced aggrecanase activity, butsubstantially no reduced activity of at least one other proteinase,where substantially no means less than 10 fold reduction, usually lessthan 5 fold reduction and in many instances less than 1 fold reduction,where activity is measured as described in Amer et al., (1999), supra.

Naturally occurring or synthetic small molecule compounds of interestinclude numerous chemical classes, though typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Also of interest as active agents are antibodies that at least reduce,if not inhibit, the target aggrecanase activity in the host. Suitableantibodies are obtained by immunizing a host animal with peptidescomprising all or a portion of the target protein, e.g. aggrecanase.Suitable host animals include mouse, rat sheep, goat, hamster, rabbit,etc. The origin of the protein immunogen may be mouse, human, rat,monkey etc. The host animal will generally be a different species thanthe immunogen, e.g. human aggrecanase used to immunize mice, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part ofaggrecanase, where these residues contain the post-translationmodifications, such as glycosylation, found on the native targetprotein. Immunogens comprising the extracellular domain are produced ina variety of ways known in the art, e.g. expression of cloned genesusing conventional recombinant methods, isolation from HEC, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the target protein, where the target proteinwill preferably be in substantially pure form, comprising less thanabout 1% contaminant. The immunogen may comprise the complete targetprotein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil & water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using aggrecanase bound to an insoluble support,protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267–73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

For in vivo use, particularly for injection into humans, it is desirableto decrease the antigenicity of the antibody. An immune response of arecipient against the blocking agent will potentially decrease theperiod of time that the therapy is effective. Methods of humanizingantibodies are known in the art. The humanized antibody may be theproduct of an animal having transgenic human immunoglobulin constantregion genes (see for example International Patent Applications WO90/10077 and WO 90/04036). Alternatively, the antibody of interest maybe engineered by recombinant DNA techniques to substitute the CH1, CH2,CH3, hinge domains, and/or the framework domain with the correspondinghuman sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

In yet other embodiments of the invention, the active agent is an agentthat modulates, and generally decreases or down regulates, theexpression of the gene encoding the target protein in the host. Forexample, antisense molecules can be used to down-regulate expression ofaggrecanase in cells. The anti-sense reagent may be antisenseoligonucleotides (ODN), particularly synthetic ODN having chemicalmodifications from native nucleic acids, or nucleic acid constructs thatexpress such anti-sense molecules as RNA. The antisense sequence iscomplementary to the mRNA of the targeted gene, and inhibits expressionof the targeted gene products. Antisense molecules inhibit geneexpression through various mechanisms, e.g. by reducing the amount ofmRNA available for translation, through activation of RNAse H, or sterichindrance. One or a combination of antisense molecules may beadministered, where a combination may comprise multiple differentsequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840–844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra, and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434–42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43–56.

As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. Generally, the desired result isat least an enhancement or reduction in aggrecanase activity, asmeasured by aggrecan cleavage product production, as compared to acontrol.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredmodulation of aggrecanase activity, e.g. desired reduction in aggrecancleavage product production. Thus, the agent can be incorporated into avariety of formulations for therapeutic administration. Moreparticularly, the agents of the present invention can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

As such, administration of the agents can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins, with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, e.g. antisense composition, it may be introduced into tissuesor host cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992),Anal Biochem 205:365–368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152–154), where gold microprojectiles arecoated with the therapeutic DNA, then bombarded into skin cells.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

The subject methods find use in the treatment of a variety of differentdisease conditions involving aggrecanase activity. Of particularinterest is the use of the subject methods to treat disease conditionscharacterized by the presence of aggrecan cleavage products,particularly 60 kDa aggrecan cleavage products having an ARGSN-terminus. Specific diseases that are characterized by the presence ofsuch methods include: rheumatoid arthritis, osteo-arthritis, infectiousarthritis, gouty arthritis, psoriatic arthritis, spondolysis, sportsinjury, joint trauma, pulmonary disease, fibrosis, and the like.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as hyperphosphatemia. Assuch, treatment also includes situations where the pathologicalcondition, or at least symptoms associated therewith, are completelyinhibited, e.g. prevented from happening, or stopped, e.g. terminated,such that the host no longer suffers from the pathological condition, orat least the symptoms that characterize the pathological condition.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

Kits with unit doses of the active agent, usually in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

The following examples are offered primarily for purposes ofillustration. It will be readily apparent to those skilled in the artthat the formulations, dosages, methods of administration, and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

EXPERIMENTAL

A nucleic acid array carrying 699 known metalloproteinase genes andnovel ESTs available in public and proprietary databases was designed.These sequences on the array were selected by a search with a seed setof known metalloprotease protein sequences from all species. Theseprotein sequences were used to find matching sequences in humannucleotide databases (GenBank and LifeSeq) at the protein (codon) level.Redundant sequences were eliminated, remaining sequences assembled andclustered, and the unique set of 699 sequences were arrayed.

The resultant array was used to screen genes expressed in primarycultures of synoviocytes and chondrocytes. A fair number ofmetalloproteinases known to be expressed by these cells were identified.However, a number of ESTs for novel proteins were also identified. Oneof these ESTs was MPS5584x1. Upon further examination, this EST wasfound to be the human homolog of the murine ADAMTS-1, which haspreviously been cloned.

From the Incyte database, by homology search, we obtained other colinearhuman ESTs corresponding to the previously cloned mouse sequence. Bysynthesizing corresponding primers, the entire human ADAMTS cDNA wassynthesized using PCR protocols. FIG. 1 provides the full lengthsequence of the human ADAMTS-cDNA (also referred to herein as humanaggrecanase). An alignment analysis of this ADAMTS cDNA with the twoADMPs reported in WO 99/05291 (seq1 and seq2) and the novelmetalloprotease reported in WO 97/31931 (w35293) was done using GAP(Needleman-Wunsch). (GAP program in GCG 10.0. A pairwise DNA alignmentwas made between each of the sequences; scoring matrix=PAM250. Gap andBestFit were originally written for Version 1.0 by Paul Haeberli from acareful reading of the Needleman and Wunsch (J. Mol. Biol. 48; 443–453(1970)) and the Smith and Waterman (Adv. Appl. Math. 2; 482–489 (1981))papers. Limited alignments were designed by Paul Haeberli and added tothe Package for Versions 3.0. They were united into a single program byPhilip Delaquess for Version 4.0).

The following results were obtained:

Pairwise comparisons using Gap (Needleman-Wunsch), Matrix PAM250. seq1seq2 w35293 adamtslhum 50% 49% 33% (similarity) 38% 39% 15% (identity)seq1 43% 28% 32% 19% seq2 35% 18%

The human ADAMTS is clearly different from seq1, seq2 and w35293.

It is apparent from the above results and discussion that humanaggrecanase, as well as polypeptides related thereto and nucleic acidcompositions encoding the same, are provided by the subject invention.These polypeptide and nucleic acid compositions find use in a variety ofdiverse applications, including research, diagnostic, screening andtherapeutic applications. Also provided are novel methods of treatingdiseases associated aggrecanase, as the identification of the subjectaggrecanase provides for an additional target for therapeutic agents forsuch diseases. Accordingly, the subject invention provides for asignificant contribution to the field.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. An isolated aggrecanase comprising the amino acid sequence set forthin SEQ ID NO:
 2. 2. An isolated aggrecanase that consists of the aminoacid sequence set forth in SEQ ID NO: 2.